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Plant Physiol. (1999) 119: 1065-1072

Embryo-Specific Gene Expression in Microspore-Derived Embryos of Brassica napus. An Interaction between Abscisic Acid and Jasmonic Acid1,2

Dirk B. Hays3, *, Ronald W. Wilen, Chuxing Sheng, Maurice M. Moloney, and Richard P. Pharis

Department of Biological Sciences, University of Calgary, Calgary, AB, Canada T2N IN4 (D.B.H., C.S., M.M.M., R.P.P.); and the Crop Development Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5A8 (R.W.W.)

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

The induction of napin and oleosin gene expression in Brassica napus microspore-derived embryos (MDEs) was studied to assess the possible interaction between abscisic acid (ABA) and jasmonic acid (JA). Napin and oleosin transcripts were detected sooner following treatment with ABA than JA. Treatment of MDEs with ABA plus JA gave an additive accumulation of both napin and oleosin mRNA, the absolute amount being dependent on the concentration of each hormone. Endogenous ABA levels were reduced by 10-fold after treatment with JA, negating the possibility that the observed additive interaction was due to JA-induced ABA biosynthesis. Also, JA did not significantly increase the uptake of [3H-ABA] from the medium into MDEs. This suggests that the additive interaction was not due to an enhanced carrier-mediated ABA uptake by JA. Finally, when JA was added to MDEs that had been treated with the ABA biosynthesis inhibitor fluridone, napin mRNA did not increase. Based on these results with the MDE system, it is possible that embryos of B. napus use endogenous JA to modulate ABA effects on expression of both napin and oleosin. In addition, JA could play a causal role in the reduction of ABA that occurs during late stages of seed development.

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Several factors in Brassica napus embryos are known to induce the expression of genes that encode the storage protein napin and the major oil-body protein oleosin. These are osmoticum and two plant hormones, ABA (Finkelstein et al., 1986; Wilen et al., 1990) and JA (Wilen et al., 1991). Whereas an interaction between NaCl and ABA and between osmoticum and ABA on embryo-specific gene expression has been documented (Finkelstein et al., 1986; Wilen et al., 1990; Bostock and Quatrano, 1992; Plant et al., 1994), the possible interaction of ABA, osmoticum, or NaCl with JA has not been investigated with regard to embryo-specific gene expression.

JA and its methyl ester, methyl jasmonate, are commonly referred to as jasmonates. They are naturally occurring plant growth regulators (Meyer et al., 1984) derived from linolenic acid in a lipoxygenase-dependent pathway (Vick and Zimmerman, 1984). JA can influence several aspects of plant growth and development, including inhibition of germination (Wilen et al., 1991, 1994), promotion of leaf abscission (Curtis, 1984) and promotion of senescence (Ueda et al., 1981). JA levels are enhanced by water stress (Creelman and Mullet, 1995) and wounding (Creelman et al., 1992b), factors that, along with applied JA, have been shown to induce the expression of genes encoding both lipoxygenases and vegetative storage proteins in soybean (Mason and Mullet, 1990; Bell and Mullet, 1991).

There is now a growing body of work that demonstrates an overlap in the biological activities of ABA and jasmonates (see Staswick, 1995). Both ABA and jasmonates can inhibit plant growth, inhibit seed germination, promote tuberization, promote senescence, and induce the expression of a number of the same genes (for review, see Staswick, 1995). Exogenous ABA and JA have also been shown to induce proteinase inhibitor II mRNA accumulation in potato (Hildmann et al., 1992), as well as seed storage and oil-body protein mRNA accumulation in B. napus (Wilen et al., 1991). There is also evidence for an additive interaction between ABA and jasmonates on inhibiting seed germination in Arabidopsis (Staswick et al., 1992), cornflower, alfalfa, cress, maize, and wheat (Wilen et al., 1994). However, in rice roots, applied JA decreased the level of expression for ABA-induced group 3 late-embryogenesis-abundant transcripts (Moons et al., 1997).

The objective of our study was to clarify the possible interaction between ABA and JA in the induction of napin and oleosin mRNA accumulation. We have used MDEs of B. napus to demonstrate that JA-induced napin and oleosin mRNA accumulation may be dependent upon endogenous ABA.

    MATERIALS AND METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Chemicals, Enzymes, and Growth Regulators

Suc for MDE medium was purchased from BDH (Poole, UK). Restriction enzymes were purchased from Pharmacia. (RS)-ABA was purchased from Sigma and JA (90% pure) from Apex Organics (STEP Centre, Osney Mead, Oxford, UK). (-) DHA was a gift from Dr. Suzanne Abrams (Plant Biotechnology Institute, Saskatoon, Canada). [2H6]-ABA was custom synthesized for R.P.P. by Drs. Martial Saugy and Laurent Rivier (Institute de Biologie et de Physiol Vegetales, Lausanne, Switzerland), [3H6]-(RS)-ABA was purchased from Amersham, and fluridone from Eli Lily (Indianapolis, IN). All plant hormones, plant hormone analogs, and plant hormone inhibitors used for treatment of the MDEs were made up in stock solutions using 50% ethanol as a solvent.

Plasmids

The Brassica napus napin cDNA clone (pN2) was obtained from Dr. Martha Crouch (University of Indiana, Bloomington, IN). The oleosin clone pOB800 was obtained from Dr. Gijs van Rooijen (University of Calgary, Alberta, Canada). The constitutive gene pGS43 was obtained from Dr. John Harada (University of California, Davis).

Plant Materials

B. napus cv Topas (seed from Dr. Keith Downey, Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada) plants were grown for 5 weeks at 25°C day/16°C night temperatures with a 16-h photoperiod (400 mmol m-2 s-1), then transferred to 12°C day/7°C night temperatures until flower buds were harvested, approximately 10 d later.

MDE Culture

MDE culture was performed as described previously (Hays et al., 1996). We treated embryos with ABA or JA by adding 10 µL of growth regulator from 10 to 100 mM stock solutions. Control embryos were treated with 10 µL of 50% ethanol. Embryos were harvested at various times after treatment. Sieving through 250-µm nylon membranes was used to harvest torpedo-stage embryos. In experiments involving the use of (-) DHA and JA, torpedo-stage MDEs were pretreated with (-) DHA (2 h) followed by addition of appropriate concentrations of JA for an additional 48 h. In experiments where fluridone was used to reduce endogenous ABA levels, the torpedo-stage MDEs were pretreated with 100 to 200 µM fluridone (Eli Lily) for 48 h followed by the addition of ABA or JA for another 48 h. Experiments with (-) DHA and fluridone were performed in triplicate.

Plasmid DNA Preparation and Oligo Labeling Reactions

Plasmid DNA was prepared according to the procedures of Sambrook et al. (1989). Once isolated, the plasmids were digested with the appropriate restriction enzymes and the inserts isolated with a commercial gene clean kit (PGC Scientific, Fredrick, MD) according to the manufacturer's protocol.

Fifty nanograms of DNA was labeled with [32P]dCTP by the random oligonucleotide-priming method (Feinberg and Vogelstein, 1984). The labeled probes were purified by applying the oligolabelling mixture to a Sephadex G50 spin column and centrifuging at 1000 rpm for 1 min. The [32P]dCTP-labeled probes were used immediately for hybridization.

RNA Extraction, Northern Hybridization, and Slot-Blot Analysis

We extracted total RNA according to the method of Verwoerd et al. (1988) using approximately 100 to 200 mg (fresh weight) of MDEs per extraction. RNA levels were quantified by UV absorption at 260 nm. Total RNA was separated by electrophoresis on 6% formaldehyde gels (1× Mops, 1.2% agarose). The RNA was then transferred to Gene Screen Plus membranes (NEN-Dupont) by capillary blotting with 20× SSC (1× SSC = 150 mM sodium chloride, 15 mM sodium citrate) for 24 h. The RNA was then fixed to the membrane by exposure to UV irradiation for 5 min. Prehybridization was carried out in Seal-a-Meal bags with 25 mL of hybridization solution (50% formamide, 5× SSPE [1× SSPE = 150 mM sodium chloride, 10 mM sodium phosphate, 1 mM EDTA], 1% SDS, and 5× Denhardt's solution) with 5 mg of yeast tRNA at 43°C for 12 h. Hybridization was carried out in fresh hybridization solution for 16 h with 5 mg of yeast tRNA and 50 ng of radiolabeled oleosin, napin, or pGS43 cDNA. Membranes were washed twice in 2× SSPE, 0.1% SDS at room temperature for 20 min, and twice in 0.2× SSPE, 0.1% SDS at 65°C for 20 min. Filters were exposed to Kodak XAR 5 film at -70°C for varying times.

For slot blots, we equalized total RNA loading using UV absorption at 260 nm and we equalized ethidium bromide-stained ribosomal bands with 6% formaldehyde gels before loading total RNA directly onto Gene Screen Plus membranes using a Minifold II slot-blot manifold (Schleicher & Schuell). We checked the loading of the RNA by hydrizing the membrane with the constitutively expressed gene pGS43 (Harada et al., 1988), as described previously (Wilen et al., 1993). Densitometry was performed on exposed x-ray films of slot blots using a scanning densitometer (Hoefer Scientific) linked to an integrator (Hewlett-Packard). Densitometry signals from exposed x-ray films were the average of the three readings. To calculate the relative induction of the napin and oleosin gene families, densitometry readings from samples containing total RNA that had been extracted from MDEs treated with 10 µM ABA were arbitrarily assigned a value of 100%. All other values were then normalized to this 100% value (Wilen et al., 1993). The signal obtained from hybridization with pGS43 was used to correct for variations in RNA loading.

Extraction and Purification of ABA

The freeze-dried MDEs (100-200 mg) were powdered in liquid N2 and extracted three times in chilled 80% aqueous methanol to which 25 to 50 ng of [2H6]-ABA and 50,000 dpm of 3H6-ABA (6 Ci mol-1) was added; the amount of [2H6]-ABA added depended on the treatment. The methanolic extract was then filtered through a Whatman no. 1 filter and the methanol phase removed in vacuo at 35°C. The aqueous extract was then adjusted to pH 3.0 with 1% acetic acid, partitioned three times against n-hexane, and then three times against water-saturated dichloromethane. The ABA, which partitioned into dicloromethane, was then taken to dryness under a gentle flow of N2. Then reversed-phase C18-HPLC on a Radial Pak liquid chromatography cartridge (10 cm × 8 mm i.d., Waters Associates) was accomplished using a gradient of 10% methanol in 1% acetic acid for the first 10 min, then a linear gradient to 73% methanol in 1% acetic acid over 30 min with a flow rate of 2 mL min-1 (Koshioka et al., 1983). Fractions corresponding to the retention times of [3H]-ABA were taken to dryness and analyzed with GC-MS-SIM.

ABA Uptake Experiments

We performed the ABA uptake experiments as described previously (Wilen et al., 1993). Four thousand torpedo-stage MDEs were suspended in 5 mL of NLN medium (Lichter, 1982) containing 100,000 dpm of [3H6]-(RS)-ABA (6 Ci mol-1) with or without 1 or 30 µM JA. The embryos were then incubated for various times at 24°C with gentle agitation (30 rpm) on a reciprocal shaker. Following 1, 4, or 18 h of incubation, embryos were separated through a coarse nylon sieve (350 µm), then washed three times with NLN medium to remove surface radiolabel. The embryos were then frozen, lyophilized, weighed, and extracted three times with 80% methanol. The purification and C18-HPLC separation of [3H]-ABA from its metabolites was carried out as described above. Purified C18-HPLC fractions that contained [3H]-ABA were quantified by liquid-scintillation counting.

GC-MS-SIM Analysis of ABA

Purified samples that were expected to contain ABA were methylated with ethereal diazomethane, taken to dryness, resuspended in n-hexane, and injected onto a DB1701-15N fused silica capillary column (15 m × 0.25 mm i.d., 0.25-µm methyl silica fill; J&W Scientific, Folsom, CA) or a DB1-15N fused silica capillary column (15 m × 0.25 mm i.d., 0.25-µm methyl silicone film; J&W Scientific) and analyzed via GC-MS-SIM. The temperature program was set for 50°C (0.1 min), then increased to 190°C at 20°C min-1, followed by 5°C min-1 to 260°C. Data analysis of ABA was carried out following the isotope dilution formula of Cohen et al. (1986).

Statistical Analysis

We used the results from individual trials (three replicate experiments performed where indicated) to calculate means. Standard errors and significant differences between treatments were determined using the Student-Newmen-Keuls test and the SPSS software package (SPSS Inc., Chicago, IL).

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Induction of Napin and Oleosin mRNA Accumulation after ABA and JA Treatment

ABA and JA are rapid and effective inducers of napin and oleosin mRNA accumulation (Figs. 1 and 2). The optimal concentration of each hormone for the induction of the genes was previously determined to be 10 µM ABA and 30 µM JA (Wilen, 1992; Hays, 1996). Thus, we used MDE cultures to determine the time course of napin and oleosin mRNA accumulation in response to exogenous ABA and JA at these concentrations. Each experiment was performed in triplicate. It is clear from Figures 1 and 2 that napin and oleosin mRNA accumulate more rapidly in response to ABA than to JA. By 48 h, however, napin mRNA levels were similar using either hormone (Fig. 1). However, JA was significantly less effective than ABA at inducing oleosin gene expression (Fig. 2).


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  		Figure 1. Napin mRNA accumulation in MDEs after treatment with 10 µM ABA and 30 µM JA for various times. Relative intensities of densitometry scans of autoradiographs from slot blots of total RNA (5 µg/slot) probed with a [32P]dCTP-labeled napin cDNA clone are plotted. Values are the mean of three experiments ± SE. The same blot was reprobed with pGS43 (Harada et al., 1989), a gene expressed constitutively in developing zygotic embryos. The signal obtained from this hybridization was used to correct for slight variations in RNA loading.


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  		Figure 2. Oleosin mRNA accumulation in MDEs after treatment with 10 µM ABA and 30 µM JA for various times. Relative intensities of densitometry scans of autoradiographs from slot blots of total RNA (5 µg/slot) probed with a [32P]dCTP-labeled oleosin cDNA clone are plotted. Values are the mean of three experiments ± SE. The same blot was reprobed with pGS43 (Harada et al., 1989), a gene expressed constitutively in developing zygotic embroys. The signal obtained from this hybridization was used to correct for slight variations in RNA loading.

Interactions between ABA and JA

To investigate the possible interactions between ABA and JA, MDEs were treated with ABA (0-10 µM) in the presence of JA (0-30 µM). Treatment with optimal levels of ABA (10 µM) and JA (30 µM) resulted in napin mRNA accumulation, which was approximately 1.5-fold greater than the accumulation of napin mRNA in response to either hormone applied alone (Fig. 3). Similarly, oleosin mRNA accumulation was much greater following application of both hormones than when 10 µM ABA or 30 µM JA was applied alone (Fig. 4). This suggests a possible additive interaction. An additive induction of oleosin mRNA accumulation was also detected when suboptimal concentrations of either hormone were used in combination (Fig. 4). Thus, when a suboptimal concentration of JA (1.0 µM) was applied with suboptimal concentrations of ABA (0.25 or 1.0 µM), napin and oleosin mRNA were detected at levels equivalent to or greater than those resulting from treatment with optimal concentrations of ABA or JA alone. However, the genes encoding oleosin responded differently to various combinations of ABA and JA concentrations, than did genes encoding napin (Figs. 3 and 4).


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  		Figure 3. Napin mRNA accumulation in MDEs after treatment with various combinations of ABA and JA for 48 h. Relative intensities of densitometry scans of autoradiographs from slot blots of total RNA (5 µg/slot) probed with a [32P]dCTP-labeled napin cDNA clone are plotted. Values are the mean of three experiments ± SE. The same blot was reprobed with pGS43 (Harada et al., 1989), a gene expressed constitutively in developing embryos. The signal obtained from this hybridization was used to correct for variations in RNA loading.


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  		Figure 4. Oleosin mRNA accumulation in MDEs after treatment with various combinations of ABA and JA for 48 h. Relative intensities of densitometry scans of autoradiographs from slot blots of total RNA (5 µg/slot) probed with a [32P]dCTP-labeled oleosin cDNA clone are plotted. Values are the mean of three experiments ± SE. The same blot was reprobed with pGS43 (Harada et al., 1989), a gene expressed constitutively in developing zygotic embryos. The signal obtained from this hybridization was used to correct for variations in RNA loading.

Effect of JA on Endogenous ABA Levels and on ABA Uptake

One possible explanation for the additive interactions between ABA and JA on embryo-specific gene expression is the possibility that applied JA may stimulate an accumulation of endogenous ABA. To test this hypothesis we determined ABA levels in MDEs by GC-MS-SIM following a 48 h incubation with applied JA (0-100 µM) (Fig. 5). Applied JA significantly (at P <=  0.05) reduced the endogenous pool of ABA by 10-fold at JA doses of 1.0 to 100 µM (Fig. 5).


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  		Figure 5. Effect of various concentrations of JA on endogenous ABA content in MDEs of B. napus. Values are the mean of three independent experiments ± SE. Embryos were treated for 48 h. Letters above each bar indicate significant groupings (P <=  0.05) via the Student-Newmen-Keuls test.

Because MDEs develop in a liquid medium and the additive interactions of ABA and JA were the result of an exogenously supplied ABA and JA, it was also important to determine whether JA had an effect on the uptake of ABA from the medium. This was done, and when [3H]-ABA uptake was normalized to the dry weight of the embryos, JA had no significant effect on the uptake of ABA from the medium (Table I).

 
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  		Table I. The effect of JA on the uptake of [3H]ABA in MDEs of B. napus

Numbers represent the mean percentage of the applied [3H](RS)-ABA (100,000 dpm) that was extracted from washed MDEs incubated for 1, 4, or 18 h. The means ± SE were calculated from triplicate assays, with an equal number of embryos present in each replicate. Because differential growth occurs in the embryos during treatment, the mean percentage of extractable [3H]ABA was also expressed when normalized to the final dry weight of the embryos. Letters (a or b) in front of the 18-h value represent significance between JA treatment within a column at (P <=  0.05) using the SNK test.

JA Action Occurs Only via ABA

Because applied JA does not yield increases in the endogenous pool of ABA, nor increase the uptake of ABA from the medium, another possible explanation is that JA may be optimizing an intermediate step in the signal transduction pathway between ABA and the induction of napin and oleosin mRNA gene expression. Such a scenario would suggest that JA requires ABA as an intermediate in the JA-induced expression of the oleosin and napin genes. To test whether ABA was required for JA-induced expression of napin, MDEs were treated with (-) DHA, a competitive inhibitor of ABA-induced gene expression (Wilen et al., 1993, 1996). Earlier, Wilen et al. (1993) showed that treatment of B. napus MDEs with (-) DHA gave a 5- to 7-fold increase in endogenous ABA levels. Thus, we pretreated MDEs with (-) DHA and then treated them with 10 or 30 µM JA. Under these conditions napin mRNA accumulation was greater than when JA was applied alone at 30 µM (Fig. 6), although at 50 µM JA, napin transcripts were not detected in the (-) DHA-pretreated MDEs (Fig. 6). Wilen (1992) observed similar results when oleosin gene expression was investigated in combination with JA and (-) DHA.


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  		Figure 6. The effect of (-) DHA alone and in combination with various concentration of JA on napin gene expression. Total RNA (20 µg lane-1) was isolated from torpedo-stage MDEs that were treated for 48 h with 10 µL 50% ethanol (control, lane C), with 30 µM JA (lane J) with 40 µM (-) DHA (lane D) or were pretreated for 2 h with (-) DHA then treated for 48 h with 10 µM (lane 10), 30 µM (lane 30), or 50 µM (lane 50) JA. These latter three samples are designated by the heading DHA + JA. This experiment was performed in triplicate. The results shown are a representative sample.

To explore the interaction between ABA and JA in more detail, we then used fluridone, an inhibitor of ABA biosynthesis (Zeevaart and Creelman, 1988). The MDEs were thus pretreated for 48 h with 200 µM fluridone. This resulted in a 4- to 7-fold reduction in endogenous ABA (to 13.0 ± 0.2 SE ng g-1 dry weight) compared with control levels (75 ± 35 ng g-1 dry weight). Then we treated MDEs with 30 µM JA or 10 µM ABA for 48 h. It was clear that the fluridone-induced reduction in endogenous ABA levels was correlated with a total loss of the ability of JA to induce napin mRNA accumulation. Fluridone treatment, however, did not inhibit napin mRNA accumulation in response to applied ABA (Fig. 7).


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  		Figure 7. Effect of fluridone on ABA- and JA-induced napin mRNA accumulation in MDEs. Values are based on densitometry scans of northern blots. All values are calculated relative to values from embryos treated with 10 µM (±)-ABA for 48 h. For fluridone treatments, MDEs were pretreated with 200 µM fluridone for 48 h, at which point fluridone-treated embryos were either maintained on the same medium or treated for an additional 48 h with ABA or JA, with fluridone still present in the medium. Stippled bars represent results from MDEs that were not treated with fluridone. This experiment was performed in triplicate. The results shown are representative.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Napin and oleosin gene expression in MDEs of B. napus can be regulated by applied ABA (Figs. 1-4) and osmoticum (Wilen et al., 1990). JA also induces the accumulation of napin mRNA (Figs. 1 and 3), but oleosin transcripts accumulate to a much lesser degree (Figs. 2 and 4). In addition, JA and ABA interact additively on embryo-specific gene expression when applied in a factorial manner (Figs. 3 and 4). Such an interaction between ABA and JA is not without precedent. For example, in rice suspension cells, Em mRNA levels approximately doubled when ABA was applied in the presence of NaCl (Bostock and Quatrano, 1992). Also, desiccation tolerance and fatty acid accumulation were enhanced in celery embryos by treatments of ABA plus Pro (Kim and Janick, 1991). For seed germination in Arabidopsis, methyl jasmonate, combined with ABA, inhibited seed germination to a greater extent (i.e. 2-fold greater) than either compound applied alone (Staswick et al., 1992). Similar results were obtained for seeds of alfalfa, cornflower, cress, maize, and wheat (Wilen et al., 1994).

There are a number of possible explanations for the interaction between ABA and JA. One is that JA may stimulate an increase in endogenous ABA levels. This was considered because Melan et al. (1993) had demonstrated that jasmonates induce lipoxygenase gene expression, and Creelman et al. (1992a) had shown that a lipoxygenase-like enzyme was involved in the biosynthesis of ABA from carotenoids. However, in our MDE system, exogenous JA had the opposite effect, with endogenous ABA levels being reduced by up to 10-fold (Fig. 5). This novel result may explain some of the variable results that have been obtained with the use of jasmonates in germination studies on a range of different species (Corbineau et al., 1988; Ranjan and Lewak, 1992; Staswick et al., 1992; Wilen et al., 1994).

Neither the reduction in endogenous ABA levels by applied JA nor the additive interaction of ABA plus JA on gene expression can be readily explained by a modulation in carrier-mediated ABA uptake by JA. That is, JA neither inhibited nor significantly increased the uptake of [3H]-ABA by the MDEs (Table I). Had JA increased the uptake, this could have explained the synergistic interaction between ABA and JA (Figs. 3 and 4). Our results with MDEs are, however, in contrast to experiments with runner-bean, cell-suspension cultures where there was a clear increase in the uptake of ABA from the medium in the presence of methyl jasmonate (Astle and Rubery, 1985).

How else might we explain how ABA and JA interact to additively induce napin and oleosin gene expression? First, JA could enhance a rate-limiting step in the transduction pathway between ABA and gene expression. Second, JA may alter the cytoplasmic concentration of ABA by decreasing the cytoplasmic pH, because ABA compartmentation appears to be pH dependent (Cowan et al., 1982; Zeevaart and Creelman, 1988). In fact, Astle and Rubery (1985) have demonstrated that reduction in cytoplasmic pH was due to exogenous MeJA. Unfortunately, localized changes in ABA concentration cannot, as yet, be measured at the subcellular level.

These alternative explanations imply that JA somehow uses ABA as an intermediate in JA-induced gene expression. The possible dependence of JA on endogenous ABA levels is further supported by both the use of (-) DHA (Fig. 6) and fluridone (Fig. 7). The ABA analog (-) DHA competitively inhibits ABA-induced gene expression and inhibits the catabolism of ABA to phaseic acid, thereby resulting in a 7-fold increase in endogenous ABA levels (Wilen et al., 1993). In the present study preincubation of MDEs with (-) DHA resulted in an increase in the sensitivity of the embryos to exogenous JA at concentrations that were both optimal and suboptimal for the induction of napin mRNA accumulation (Fig. 6). However, there was no gene expression when higher concentrations of JA were used in combination with (-) DHA. This is puzzling, but not unprecedented. In studies on cornflower germination, the application of JA in combination with 1 or 10 µM ABA increased the inhibition of seed germination beyond that observed with the use ABA alone. In contrast, when 30 µM ABA was applied with JA, the percent of germination actually increased (Wilen et al., 1994). In rice roots when 5 or 10 µM JA was applied in conjunction with 10 or 20 µM ABA, a salt-inducible transcript (salT) accumulated to higher levels than with ABA alone (Moons et al., 1997). However, Moons et al. (1997) found that the same transcript was reduced when a higher concentration of ABA (40 µM) was applied with 10 µM JA.

Based upon the additive induction of gene expression by the application of JA and ABA to MDEs (Figs. 3 and 4) and upon the ability of JA to reverse the effects of a competitive inhibitor of ABA (Fig. 6), it appears that JA reduces the effective concentration of ABA (endogenous or applied) that is needed to obtain maximal ABA-induced gene expression. These results imply that JA acts to increase the "sensitivity" of the embryo system to ABA (i.e. sensitivity to the ABA present in the embryo). This may occur not only at the level of gene expression, but also at the level of ABA catabolism (Fig. 5). Our results further imply that the induction of gene expression by JA is dependent on ABA as an intermediate, a conclusion supported by the use of fluridone. Preincubation of MDEs with 200 µM fluridone, a treatment that reduced endogenous ABA by 78%, eliminated JA-induced napin mRNA accumulation (Fig. 7).

The dependence of ABA as an intermediate in JA responses is also supported by the work of Staswick et al. (1992), who showed that Me-JA, which alone was not capable of inhibiting germination of Arabidopsis, could increase the sensitivity of the seeds to exogenous ABA. There also appears to be a temperature interaction. Wilen et al. (1994) showed that JA was unable to inhibit seed germination in three of the five species investigated when the germination assays were conducted at room temperature. However, when the temperature was reduced to 10°C, JA became an effective germination inhibitor. Low temperature has been shown to increase endogenous ABA levels in a variety of different species (Daie et al., 1981; Dorffling et al., 1990). The dependence of JA on ABA as an intermediate might also explain the lag in mRNA accumulation when exogenous JA is applied alone, versus the more rapid mRNA accumulation when ABA is applied alone (Figs. 1 and 2). That is, JA may first exact a positive influence on the signal transduction pathway between ABA and the induction of napin and oleosin mRNA accumulation.

It has been well demonstrated that an overlap exists between the effects of applied ABA and the effects of osmoticum, not only at the gene-expression level, but also in the inhibition of germination. It would thus be useful to determine if endogenous JA levels were modified in response to osmotic stress. For example, if endogenous JA levels were also elevated by osmotic stress, it could help to explain why applied ABA and osmotic stress yield synergistic responses (Bostock and Quatrano, 1992; Plant et al., 1994). An increase in endogenous JA levels in response to osmoticum could also help to explain the additive interaction between ABA and sorbitol in GUS expression assays of an embryo-specific oleosin promoter in B. napus (Plant et al., 1994). Because an increase in JA after dehydration has been reported in leaves of soybean (Creelman and Mullet, 1995), it seems quite reasonable to suggest that a similar increase may occur in response to osmotic stress.

The ability of JA to elicit the expression of the storage product gene napin appears to be dependent on the endogenous concentration of ABA. If so, this could have important implications with regard to a putative role for JA in regulating the rate of the maturation and seed drying process. During the late stages of seed development in many species, ABA levels have been shown to decline through to the dry seed stage (Black, 1991). If our results using the MDE system are applicable to the seed in situ, then JA may play a role in the reduction of ABA concentration in the seed, thereby allowing the seed to move expeditiously from ABA-mediated processes into late seed development under both nonstress and stress conditions.

    FOOTNOTES
3   Present address: U.S. Department of Agriculture, Agricultural Research Service, Plant Science and Entomology Research Unit, Department of Agronomy, Throckmorton Hall, Kansas State University, Manhattan, KS 66506-5502.
*   Corresponding author; e-mail dhays{at}pswcrl.ars.usda.gov; fax 1-785-532-6167.

   Received October 29, 1998; accepted December 11, 1998.
1   This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada to R.P.P. and M.M.M.
2   This paper is dedicated to Dr. Chuxing Sheng, a Ph.D. graduate in R.P.P.'s laboratory. Dr. Sheng died of cancer in August 1996.

    ABBREVIATIONS

Abbreviations: (-) DHA, (-) 2',3' dihydroacetylenic abscisyl alcohol. GC-MS-SIM, GC-MS-selective ion monitoring. JA, jasmonic acid. MDE(s), microspore-derived embryo(s).

    LITERATURE  CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References

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